Centrosome cohesion is regulated by a balance of kinase and phosphatase activities

2001 ◽  
Vol 114 (20) ◽  
pp. 3749-3757 ◽  
Author(s):  
Patrick Meraldi ◽  
Erich A. Nigg
Keyword(s):  
Protein Phosphatase ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Microtubule Depolymerization ◽  
Drug Induced ◽  
Microtubule Network ◽  
Similar Phenotype ◽  
Underlying Mechanisms

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1α (PP1α) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1α as critical regulators of centrosome structure.

scholarly journals Role of PKR in the Inhibition of Proliferation and Translation by Polycystin-1

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yan Tang ◽  
Guang Shi ◽  
JungWoo Yang ◽  
Wang Zheng ◽  
Jingfeng Tang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Synthesis ◽  
Kinase Activity ◽  
Protein Translation ◽  
Dominant Negative ◽  
Inhibition Of Proliferation ◽  
Proliferation And Apoptosis ◽  
Autosomal Dominant Polycystic Kidney ◽  
Underlying Mechanisms

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations in the PKD1 (~85%) or PKD2 (~15%) gene which, respectively, encode polycystin-1 (PC1) and polycystin-2 (PC2). How PC1 regulates cell proliferation and apoptosis has been studied for decades but the underlying mechanisms remain controversial. Protein kinase RNA-activated (PKR) is activated by interferons or double-stranded RNAs, inhibits protein translation, and induces cell apoptosis. In a previous study, we found that PC1 reduces apoptosis through suppressing the PKR/eIF2α signaling. Whether and how PKR is involved in PC1-inhibited proliferation and protein synthesis remains unknown. Here we found that knockdown of PKR abolishes PC1-inhibited proliferation and translation. Because suppressed PKR-eIF2α signaling/activity by PC1 would stimulate, rather than inhibit, the proliferation and translation, we examined the effect of dominant negative PKR mutant K296R that has no kinase activity and found that it enhances the inhibition of proliferation and translation by PC1. Thus, our study showed that inhibition of cell proliferation and protein synthesis by PC1 is mediated by the total expression but not the kinase activity of PKR, possibly through physical association.

scholarly journals MITOCHONDRIA AS REVERSIBLE REGULATORS OF AGING ASSOCIATED SKIN WRINKLES AND HAIR LOSS IN MICE

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S395-S395
Author(s):  
Keshav K Singh
Keyword(s):  
Mouse Model ◽  
Hair Loss ◽  
Transgene Expression ◽  
Skin Aging ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dominant Negative Mutation ◽  
Polymerase Domain ◽  
Skin Wrinkles

Abstract To evaluate the consequences of the decline in mtDNA content associated with aging we have created an inducible mouse model expressing, in the polymerase domain of POLG1, a dominant-negative mutation that induces depletion of mtDNA. We utilized this inducible mouse model to modulate mitochondrial function by depleting and repleting the mtDNA content. We demonstrate that, in mice, ubiquitous expression of dominant-negative mutant POLG1 leads to 1) reduction of mtDNA content in skin, 2) skin wrinkles, and 3) hair loss. By turning off the mutant POLG1 transgene expression in the whole animal, the skin and hair phenotypes revert to normal after repletion of mtDNA. Thus, we have developed whole-animal mtDNA depleter-repleter mice. These mice present evidence that mtDNA homeostasis is involved in skin aging phenotype and loss of hair and provide an unprecedented opportunity to create tissue-specific mitochondrial modulation to determine the role of the mitochondria in a particular tissue.

scholarly journals Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

1998 ◽  
Vol 143 (6) ◽  
pp. 1505-1521 ◽  
Author(s):  
Brian Storrie ◽  
Jamie White ◽  
Sabine Röttger ◽  
Ernst H.K. Stelzer ◽  
Tatsuo Suganuma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Protein Synthesis Inhibitors ◽  
Microtubule Depolymerization ◽  
Golgi Stack ◽  
Gradual Accumulation ◽  
Green Fluorescent ◽  
Er Export

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

scholarly journals Foxl2 Up-Regulates Aromatase Gene Transcription in a Female-Specific Manner by Binding to the Promoter as Well as Interacting with Ad4 Binding Protein/Steroidogenic Factor 1

2007 ◽  
Vol 21 (3) ◽  
pp. 712-725 ◽  
Author(s):  
De-Shou Wang ◽  
Tohru Kobayashi ◽  
Lin-Yan Zhou ◽  
Bindhu Paul-Prasanth ◽  
Shigeho Ijiri ◽  
...  
Keyword(s):  
Serum Levels ◽  
Sex Reversal ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Testicular Development ◽  
Aromatase Gene ◽  
Ovarian Differentiation ◽  
Forkhead Domain ◽  
17Β Estradiol

Abstract Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17β-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17β-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.

Kinase Activity of RTKs Is Dispensable for Factor Independent Growth and Transformation of a Myeloid Cell Line 32D in the Presence of Cbl Mutants.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3970-3970
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Cell Line ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Dominant Negative ◽  
Class Iii ◽  
Primary Resistance

Abstract Abstract 3970 Poster Board III-906 Purpose The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. Results We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We recently identified the first c-Cbl mutation in human disease in an AML patient, named Cbl-R420Q. Co-expression of two different dominant negative mutants of c-Cbl (Cbl-R420Q or Cbl-70Z) with Kit induced cytokine-independent proliferation, survival and clonogenic growth. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on RTKs, but independent of their kinase activity. Instead, transformation appeared to depend on Src family kinases (SFKs), as c-Cbl co-immunoprecipitated with SFKs and SFK inhibition abolished transformation. Conclusion Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. They demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML. Furthermore, these findings may explain primary resistance to tyrosine kinase inhibitors targeted at RTKs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals p130Cas mediates the transforming properties of the anaplastic lymphoma kinase

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3907-3916 ◽  
Author(s):  
Chiara Ambrogio ◽  
Claudia Voena ◽  
Andrea D. Manazza ◽  
Roberto Piva ◽  
Ludovica Riera ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Anaplastic Lymphoma Kinase ◽  
Kinase Activity ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Lymphoid Cells ◽  
Src Tyrosine Kinase ◽  
Anaplastic Lymphoma ◽  
Proteomic Approach

Translocations of the anaplastic lymphoma kinase (ALK) gene have been described in anaplastic large-cell lymphomas (ALCLs) and in stromal tumors. The most frequent translocation, t(2;5), generates the fusion protein nucleophosmin (NPM)–ALK with intrinsic tyrosine kinase activity. Along with transformation, NPM-ALK induces morphologic changes in fibroblasts and lymphoid cells, suggesting a direct role of ALK in cell shaping. In this study, we used a mass-spectrometry–based proteomic approach to search for proteins involved in cytoskeleton remodeling and identified p130Cas (p130 Crk-associated substrate) as a novel interactor of NPM-ALK. In 293 cells and in fibroblasts as well as in human ALK-positive lymphoma cell lines, NPM-ALK was able to bind p130Cas and to induce its phosphorylation. Both of the effects were dependent on ALK kinase activity and on the adaptor protein growth factor receptor–bound protein 2 (Grb2), since no binding or phosphorylation was found with the kinase-dead mutant NPM-ALKK210R or in the presence of a Grb2 dominant-negative protein. Phosphorylation of p130Cas by NPM-ALK was partially independent from Src (tyrosine kinase pp60c-src) kinase activity, as it was still detectable in Syf-/- cells. Finally, p130Cas-/- (also known as Bcar1-/-) fibroblasts expressing NPM-ALK showed impaired actin filament depolymerization and were no longer transformed compared with wild-type cells, indicating an essential role of p130Cas activation in ALK-mediated transformation.

scholarly journals Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

2001 ◽  
Vol 12 (7) ◽  
pp. 2171-2183 ◽  
Author(s):  
Juan Ángel Fresno Vara ◽  
Ma Aurora Domı́nguez Cáceres ◽  
Augusto Silva ◽  
Jorge Martı́n-Pérez
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinases ◽  
Cellular Proliferation ◽  
Tyrosine Phosphatase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Herbimycin A ◽  
Serine Kinases ◽  
Dependent Cell

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [3H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, andodc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.

Essential roles of IκB kinases α and β in serum- and IL-1-induced human VSMC proliferation

2000 ◽  
Vol 278 (6) ◽  
pp. H1823-H1831 ◽  
Author(s):  
Sebastian Sasu ◽  
Debbie Beasley
Keyword(s):  
Smooth Muscle ◽  
Fluorescent Protein ◽  
Interleukin 1 ◽  
Fold Increase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mutant Form ◽  
Mobility Shift ◽  
Vsmc Proliferation

Interleukin-1 (IL-1) is a potent vascular smooth muscle cell (VSMC) mitogen, which can stimulate cells via activation of nuclear factor-κB (NF-κB) following phosphorylation of its inhibitory subunit (IκB). Because the proliferative effect of IL-1 is additive with that of serum, the present studies assessed the role of IκB kinases (IKKs) and NF-κB in both IL-1- and serum-induced VSMC proliferation. IL-1β (1 ng/ml) induced marked and persistent NF-κB activation in VSMC that was maximal at 1 h and persisted for 3 days. There was a 3-fold increase in DNA synthesis after acute IL-1 exposure (24–96 h) and a 12-fold increase after chronic IL-1 exposure (>7 days). Electrophoretic mobility shift assay and supershift analysis indicated that IL-1-induced NF-κB complexes consisted of p65/p50 heterodimers and p50 homodimers. Human saphenous vein smooth muscle cells (HSVSMC) were transiently cotransfected with expression plasmids encoding a dominant negative mutant form of either IKKα or IKKβ, in which K44 was mutated to A (K44A), and a green fluorescent protein expression plasmid that allows identification of transfected cells. IL-1 induced nuclear localization of p65 in 95% of cells transfected with vector alone but in only 69% and 26% of cells expressing IKKα (K44A) or IKKβ (K44A), respectively. Likewise, proliferation increased 3.2-fold in IL-1-treated HSVSMC which had been transfected with vector alone, but only 2.2- and 1.5-fold proliferation in HSVSMC expressing IKKα (K44A) or IKKβ (K44A), respectively. Although serum activated NF-κB transiently, serum-induced proliferation was markedly attenuated in HSVSMC expressing IKKα (K44A) and IKKβ (K44A) compared with HSVSMC transfected with vector alone. The results support an essential role of IKKs in the proliferative response of HSVSMC to IL-1 and to serum.

scholarly journals An Essential Role of the Jak-2/STAT-3/Cytosolic Phospholipase A2Axis in Platelet-derived Growth Factor BB-induced Vascular Smooth Muscle Cell Motility

2004 ◽  
Vol 279 (44) ◽  
pp. 46122-46128 ◽  
Author(s):  
Indira Neeli ◽  
Zhimin Liu ◽  
Nagadhara Dronadula ◽  
Z. Alex Ma ◽  
Gadiparthi N. Rao
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Dominant Negative ◽  
Platelet Derived Growth Factor ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Cytosolic Phospholipase ◽  
Negative Mutant

Platelet-derived growth factor-BB (PDGF-BB) is a potent motogen for vascular smooth muscle cells (VSMCs). To understand its motogenic signaling events, we have studied the role of the Janus-activated kinase/signal transducers and activators of transcription (Jak/STAT) pathway and cytosolic phospholipase A2(cPLA2). PDGF-BB stimulated tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner in VSMCs. In addition, AG490 and Jak-2KEpRK5, a selective pharmacological inhibitor and a dominant negative mutant, respectively, of Jak-2, attenuated PDGF-BB-induced STAT-3 tyrosine phosphorylation and its DNA binding and reporter gene activities. PDGF-BB induced VSMC motility in a dose-dependent manner with a maximum effect at 10 ng/ml. Dominant negative mutant-dependent suppression of Jak-2 and STAT-3 blocked PDGF-BB-induced VSMC motility. PDGF-BB induced the expression of cPLA2in a Jak-2/STAT-3-dependent manner, and pharmacological inhibitors of cPLA2prevented PDGFBB-induced VSMC motility. Furthermore, either exogenous addition of arachidonic acid or forced expression of cPLA2rescued PDGF-BB-induced VSMC motility from inhibition by blockade of Jak-2 and STAT-3 activation. Together, these results for the first time show that PDGF-BB-induced VSMC motility requires activation of the Jak-2/STAT-3/cPLA2signaling axis.

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